The increasing number and diversity of plants containing novel traits derived from recombinant DNA research present both environmental and commercial concerns. The concerns arise from the potential for novel traits to spread by pollen to sexually compatible plants in a natural or cultivated population.
Plants with new and altered traits imparted by genetic technologies and recombinant DNA technology in particular are now viewed as the cornerstone of the crop biotechnology industry. Currently a considerable number of crops plants with novel traits that originated from tissue culture, somatoclonal variation or mutation as well as genetic engineering are undergoing field trials and the first stages of commercial release. These plants not only include conventional crops grown on an annual basis, but other plants such as trees or shrubs which comprise novel traits and are perennial in nature.
Modern crop varieties comprise both individual genes that confer a particular trait and combination of genes assembled through conventional plant breeding. Accordingly, as more novel traits are developed and incorporated into modern crop varieties, it is valuable to have a means to preserve genetic compositions, including those of specific crop varieties, cultivars or breeding lines. Of particular value is the preservation of crops which carry traits not usually found in the crop; for example, plants which produce novel oil, meal or other components or those plants modified to produce speciality chemicals. Additionally, perennial plants such as trees are being produced which carry novel traits such as altered lignin levels, insect and fungal resistance and herbicide tolerance.
Novel traits are introduced into plants by conventional breeding or genetic engineering. However, to date neither route provides features that can be routinely used for maintaining germplasm purity, or controlling persistence or potential spread of the novel trait. Current vectors and genetic compositions typically do not address two important issues: (1) commercial issues such as the prevention of transformed crop plants or elite varieties from contaminating other commercial productions, or the prevention of introgression of alien germplasm from closely related cultivars or plant species, and; (2) environmental issues such as the removal of transformed crop plants or related species that have acquired the genes in question from non-agricultural environments. Additionally current transformation methods do not provide the means for reducing the introduction of genes via pollen mediated out-crossing to other cultivars or related species (either wild or cultivated).
The single largest immediate risk for the use of many crops with novel traits is the risk of contamination among commercial productions of the same crop species. The risk of a crop species such as oilseed rape or canola (Brassica napus) to become a weed or to cross with wild weedy relatives is modest compared with the near certainty of crossing with other commercial productions of canola, especially where large production areas exist. In the past this has not been a significant problem for farmers and commercial processors for several reasons. First, breeding objectives have been relatively uniform for canola crop; second, only a small number of cultivars have comprised 90-100% of the total acreage grown by farmers; and third, the only speciality type, traditionally cultivated, high erucic acid industrial oil cultivars have been grown in physical isolation. Accordingly, cross contamination of food quality canola varieties with genes conferring high erucic acid has not been a serious issue.
Recently additional unique varieties have been released. These include varieties that carry recombinant genes which confer tolerance to herbicides and varieties developed by conventional breeding which have variations in fatty acid profile, such as high oleic acid. Purity of seed, both during production and harvesting of canola seed for crushing and processing is now a growing issue. Because of the impending modification of canola with numerous additional recombinant genes that impart different properties to the oil (e.g. high laurate content) or the use of plants as producers of heterologous proteins such as pharmaceuticals, potentially serious industrial cross contamination may be anticipated.
These issues extend to many crops in addition to Brassica oilseeds. In maize, increasing emphasis on herbicide tolerance, insect resistance and diversification of modified end products (eg. starch, oil, meal) clearly indicates that many different traits will be incorporated in the corn crop. As some maize varieties are destined for specialized use, such as wet milling or feed, or even production of pharmacologically important proteins, the issue of segregation of these speciality types from the mainstream is relevant. Considering that corn pollen can sometimes travel significant distances, a genetic means to control pollination is be highly advantageous.
Similarly, the proximity of perennial plants to their wild relatives is a problem. For instance, a transgenic tree expressing insect tolerance could cross with a wild species of tree to create a hybrid that expresses insect tolerance. Under managed conditions such as plantations, insect resistance would not have a significant environmental impact. However, should the insect resistance trait become widespread in a natural forest population a serious ecological problem could result. Insect populations are part of the food chain in a forest system and reduced levels of insects could lead to a collapse of the predator population, which is often native bird species. Accordingly, for unmanaged systems control of the spread of genes that may carry environmental consequences is a highly desirable goal.
Currently physical isolation combined with border rows that function as pollen traps have been employed to contain transgenic plants under study and development. This method, however, is impractical for widespread cultivation. Moreover, with increasing production and distribution of an increasing number of different transgenic types, the potential for contamination increases dramatically. This issue has recently become a major concern for the oilseed rape industry and will become a greater issue for other major crops (eg. corn) as the numbers of different recombinant and speciality genotypes reach the market place.
In addition to cross-contamination among commercial crop productions, another concern is the potential spread of crops used as vehicles for producing heterologous proteins of commercial or medicinal value. These novel protein products can potentially contaminate plants destined for food use and export. Although production standards can be implemented that will attempt to preserve the identity of individual transgenic lines and reduce unintended contaminations, the outflow of genes to other cultivars will eventually occur. The potential spread of genes that cannot be easily identified, e.g. by herbicide tolerance, nor impart a distinctive morphology has yet to be addressed by government or industry.
Methods which control the spread of transgenes into the environment or other commercial cultivars are also useful for preventing the introgression of alien germplasm into identity-preserved commercial varieties. In this regard “alien germplasm” is defined as any germplasm which does not comprise the full complement of traits of the identity-preserved cultivar. Accordingly alien germplasm can include both sexually compatible wild relatives and other commercial varieties of the crop. With an increasing number of plants carrying novel traits being contemplated for commercial production, methods that prevent the contamination of both seed production and commodity production will provide a valuable means to maintain germplasm purity and identity preservation.
As an example, many enzymes have been tested that alter plant oil production in oilseed crops such as soybean corn and canola. The same plant species have been used for producing inedible short chain or long chain industrially fatty acids as well as edible oil. Since modified oil seeds must be isolated to ensure pollen carrying the oil modification genes does not contaminate edible oil variety seeds, this poses a growing problem for the seed production industry. The isolation distances routinely practiced in seed production for many crops may not be sufficient to ensure required levels of purity. Where crop plants are used to produce speciality products such as pharmaceutically active compounds, even minor contamination of germplasm is highly undesirable.
Oil seed crops such as canola typically shatter seed before harvest. This results in significant numbers of volunteer plants in subsequent years, potentially contaminating subsequent commercial productions both by crossing and by direct effects of the pollen on developing grain (xenia effects). In addition, seeds retained and distributed by farmers for future planting could contribute to contamination problems.
For perennial plants, the long life of trees and the presence of indigenous wild relatives raise additional concerns. Some trees take many years to flower, producing enormous amounts of pollen that can last for many years and are especially suited for widespread wind pollination. Transgenic trees therefore pose special problems and may require mechanisms to control gene flow to wild relatives.
It has been suggested that some new crop types, through hybridization with wild relatives, may invade natural ecosystems. This and related issues have been extensively debated (eg. University of California, Risk assessment in agricultural biology: proceedings of an international conference, 1990, Casper, R., & Landsman, J., 1992, The bio-safety results of field tests of genetically modified plants and microorganisms. Proceedings of the 2nd International Symposium on The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, 1992 Goslar, Germany, Dale, P. et al., 1992, The field release of transgenic plants. The British Crop Protection Council. Brighton Crop Protection Conference: Pests and Diseases, Vols. I, II and III., Proceedings of the 3rd International Symposium on The BioSafety Results of Field Tests of Genetically Modified Plants and Microorganisms, 1994, Monterey, Calif., D. D. Jones, 1994).
The consensus of these studies and experimental results achieved to date support the view that the degree of potential spread of transgenes to wild relatives is highly dependent upon the species and environmental conditions. Crossing with relatives is not likely with some species and probable for others (Raybould & Grey, J. Applied Ecology 30: 199-219, 1993). Many crops are highly specialized and adapted to non-competitive cultivation practices and thus are not generally considered a serious environmental risk on their own (Dale et al., Plant Breeding 111:1-22, 1993, Fishlock, D., The PROSAMO Report, published by the Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, UK TW11 0LY) The potential for environmental problems due to, for example, the inclusion of a virus coat protein gene that has potential for viral recombination and the evolution of new viruses with an extended host range, is currently unknown (Gal S., et al., Virology 187:525-533, Grimsley, N., et al., EMBO Journal 5: 641-646, 1986, Lecoq, H., et al., Molec. Plant Microbe Interact. 6:403-406, 1993. Tepfer, M., Biotechnology 11: 1125-1132. 1993). Accordingly there is a need for methods to restrict the potential flow of this type of genes or to selectively eliminate those plants which contain such genes.
Attempts have been made to develop methods to specifically remove or identify plants that contain novel traits introduced by recombinant DNA. For example, the use of a conditionally lethal gene, i.e. one which results in plant cell death under certain conditions, has been suggested as a means to selectively kill plant cells containing a specific recombinant DNA. Recently the development of genes which are conditionally lethal in plants have been described (eg WO 94/03619). However, methods using these genes have been restricted to the application of a substance that triggers the expression of the lethal phenotype. For widespread agricultural practices, these methods have serious limitations.
An example of a conditionally lethal gene is the Agrobacterium Ti plasmid-derived oncogene commonly referred to as “gene 2” or “oncogene 2”. The gene encodes the enzyme indole acetamide hydrolase (IAMH) that hydrolyzes indole acetamide, a compound that has essentially no phytohormone activity, to form the active auxin phytohormone indole acetic acid. The enzyme IAMH is capable of hydrolyzing a number of indole amide substrates including naphthalene acetamide, resulting in the production of the well known synthetic plant growth regulator naphthalene acetic acid (NAA). Use of the IAMH gene for roguing plants has been described by Jorgenson (U.S. Pat. No. 5,180,873). The method requires application of NAM to discriminate plants which carry the conditionally lethal gene.
Other enzymes may also be used as conditionally lethal genes. These include enzymes which act directly to convert a non-toxic substance to a toxin, such as the enzyme methoxinine dehydrogenase, which converts non-toxic 2-amino-4-methoxy-butanoic acid (methoxinine) to toxic methoxyvinyl glycine (Margraff, R., et al., 1980, Experimentia 36: 846), the enzyme rhizobitoxine synthase, which converts non-toxic 2-amino-4-methoxy-butanoic acid to toxic 2-amino-4-[2-amino-3-hydroxypropyl]-trans-3-butanoic acid (rhizobitoxine) (Owens, L. D, et al., 1973, Weed Science 21: 63-66), the de-acylase enzyme which acts specificlly to convert the inactive herbicide derivative L-N-acetyl-phosphinothricin to the active phytotoxic agent phosphinothricin (Bartsch, K. and Schultz, A., EP 617121), and the enzyme phosphonate monoester hydrolase which can hydrolyze inactive ester derivatives of the herbicide glyphosate to form the active herbicide (Dotson S. B., and Kishore G. M., 1993, U.S. Pat. No. 5,254,801). Other conditionally lethal genes may be engineered from lethal genes. A lethal gene which is expressed only in response to environmental or physiological conditions is lethal under those conditions. For example, a gene that encodes a lethal activity may be placed under the control of a promoter that is induced in response to a specific chemical trigger or an artificial or naturally occurring physiological stress. In this fashion the expression of the lethal gene activity is conditional on the presence of the inducer.
The expression of the conditionally lethal gene that acts on a non-toxic substance to convert said substance to a toxic substance is typically regulated by a promoter that is a constitutive promoter expressed in all or most cell types or a developmentally regulated promoter expressed in certain cell types or at certain stages of development. Any promoter that provides sufficient level of expression can be used. However, in practice promoters that provide high levels of expression for extended periods offer the best opportunities to remove unwanted plants.
The need to apply a chemical to induce the lethal phenotype reduces the utility of a conditionally lethal gene. The widespread application of chemicals may be impractical and raise additional environmental concerns. Accordingly the use of conditionally lethal genes as currently described is not ideally suited for general applications since intervention is required to express the lethal phenotype.
The possibility of using a repressed lethal gene to limit the persistence of hybrid crops has been suggested recently by Oliver et al (patent application WO 96/04393). In this system expression of a lethal gene is blocked by a genetic element that binds a specific repressor protein. The repressor protein is the product of a repressor gene typically of bacterial origin. The genetic element that binds the repressor protein is referred to as a blocking sequence and is constructed such that it further comprises DNA sequences recognized by a DNA recombinase enzyme (e.g. the CRE enzyme). Plants that contain said blocked lethal gene are hybridized with plants comprising the DNA recombinase gene. Either the lethal gene or the recombinase enzyme (or both) is under control of regulatory elements that allow expression only at a specific stage of plant development (e.g. seed embryo). Consequently, the recombinase function in the resulting F1 hybrid plant removes the specific blocking sequence and activates the lethal gene so that no F2 plant is produced. Notably, this scheme cannot control outcrossing of germplasm that carries the novel trait nor introgression of alien germplasm. The method does not apply to self- or open-pollinating varieties. Accordingly, the method is useful only as a means to restrict use (e.g. re-planting) to F1 hybrid seed.
Methods to eliminate recombinant DNA sequences used to obtain transformants such as selectable markers have been developed. Use of a transposase or recombinase to remove selected recombinant sequences from transgenic crop plants has been described in U.S. Pat. No. 5,482,852 (Biologically Safe Transformation System, by Yoder and Lassner). This invention describes a method for removing vector and marker gene sequences by enclosing them within a transposon. The sequences are subsequently removed by crossing the plant to a plant with transposase function.
No published method, however, addresses the problem of contamination of related varieties by cross pollination. The art also does not provide a means to prevent the introgression of alien germplasm by pollination with related pollen, even pollen from the same variety but lacking the genetic trait(s).
Therefore, a method that limits outcrossing and introgression without intervention is needed for management and control of novel traits and crops with novel traits. A mechanism to control cross-contaminations among commercial crops is also needed. Such a mechanism is also needed in the management of perennial crops such as trees, shrubs and grapevines. In particular any mechanism which does not require intervention in order to function is ideally suited for perennial crops. The present invention describes methods and genetic compositions which respond to these needs.